Growth Kinetics of Coliform Bacteria under Conditions Relevant to Drinking Water Distribution Systems

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1991, p /91/ $02.00/0 Copyright C 1991, Americn Society for Microbiology Vol. 57, No. 8 Growth Kinetics of Coliform Bcteri under Conditions Relevnt to Drinking Wter Distribution Systems ANNE K. CAMPER,'* GORDON A. McFETERS,2 WILLIAM G. CHARACKLIS,1 AND WARREN L. JONES' Center for Interfcil Microbil Process Engineering' nd Deprtment of Microbiology,2 Montn Stte University, Bozemn, Montn Received 15 Jnury 1991/Accepted 17 My 1991 The growth of environmentl nd clinicl coliform bcteri under conditions typicl of drinking wter distribution systems ws exmined. Four coliforms (Klebsiell pneumonie, Escherichi coli, Enterobcter erogenes, nd Enterobcter cloce) were isolted from n operting drinking wter system for study; n enterotoxigenic E. coli strin nd clinicl isoltes of K. pneumonie nd E. coli were lso used. All but one of the coliforms tested were cpble of growth in unsupplemented minerl slts medium; the environmentl isoltes hd greter specific growth rtes thn did the clinicl isoltes. This trend ws mintined when the orgnisms were grown with low levels (<1 mg liter-') of yest extrct. The environmentl K. pneumonie isolte hd greter yield, higher specific growth rtes, nd lower Ks vlue thn the other orgnisms. The environmentl E. coli nd the enterotoxigenic E. coli strins hd comprble yield, growth rte, nd Ks vlues to those of the environmentl K. pneumonie strin, nd ll three showed significntly more successful growth thn the clinicl isoltes. The environmentl coliforms lso grew well t low tempertures on low concentrtions of yest extrct. Unsupplemented distribution wter from the collborting utility supported the growth of the environmentl isoltes. Growth of the K. pneumonie wter isolte ws stimulted by the ddition of utoclved biofilm but not by tubercle mteril. These findings indicte tht growth of environmentl coliforms is possible under the conditions found in operting municipl drinking wter systems nd tht these bcteri could be used in tests to determine ssimilble orgnic crbon in potble wter. Occurrences of coliforms in finished drinking wter in the bsence of known breches of tretment brriers continue to be mjor problem in the drinking wter industry nd hve emerged s criticl regultory issue. Coliforms my grow within the distribution system t the expense of orgnic crbon. Sufficient quntities of other nutrients (nitrogen, phosphorus) must lso be vilble in the wter to support the prolifertion of suspended or biofilm coliforms. Levels of bcteril nutrients present in potble wter re typiclly low, with totl orgnic crbon concentrtions in finished drinking wter vrying from 0.05 to 12.2 mg liter-1 (9, 10). The microbilly ssimilble frction is even smller, rnging from 3 to 500,ug liter-' (2, 9). However, these concentrtions of orgnic mteril in potble wter hve been shown to support the growth of vrious heterotrophic orgnisms nd some coliforms (1, 9, 11-13). Since coliforms re trditionlly viewed s copiotrophic, especilly fecl biotypes, experiments were performed to determine whether coliform bcteri originting from drinking wter distribution system were cpble of growth under typicl temperture nd nutrient concentrtions. Prior reserch on coliform bcteri isolted from drinking wter distribution system demonstrted tht no one phenotypic chrcteristic could differentite between environmentl nd clinicl isoltes of the sme species (5), but growth rtes hve not been previously determined. In the experiments reported herein we used coliform bcteri isolted from the New Hven, Conn., drinking wter distribution system, where unexplined coliform occurrences hve been reported (3). Comprisons of growth rtes nd cell yields t vrious tempertures nd nutrient concentrtions were mde with clinicl isoltes of the sme * Corresponding uthor species. These dt were used to ssess the specific cpbility of environmentlly isolted coliforms for growth under conditions relevnt to distribution systems. This informtion is importnt from regultory stndpoint, since the presence of coliforms is used s n indictor of potble wter qulity. In ddition, n enterotoxigenic Escherichi coli (ETEC) strin ws used to determine whether known pthogen ws cpble of repliction under the low-nutrient nd low-temperture conditions representtive of those in drinking wter. Coliforms isolted from nd presumed to be proliferting in drinking wter distribution system my be considered by some to be unimportnt from public helth stndpoint, but growth of known enteric pthogens under these conditions cnnot be disregrded. Experiments were lso performed to evlute potentil nutrient sources in the New Hven wter, including biofilm, pulverized tubercle, nd unsupplemented drinking wter. Determintion of opertionl prmeters leding to the prolifertion of these orgnisms my ssist utilities in selecting nd implementing pproprite remedil ctions. MATERIALS AND METHODS Orgnisms. Environmentl strins of E. coli, Klebsiell pneumonie, Enterobcter cloce, nd Enterobcter erogenes were isolted during routine monitoring of the municipl drinking wter distribution system t New Hven. These orgnisms were ssocited with unexplined occurrences of coliforms in finished wter. The ETEC strin H10407 (078: Hll) ws obtined from R. A. Wilson, E. coli Reference Center, Pennsylvni Stte University, University Prk. A clinicl isolte of K. pneumonie ws procured from the Montn Stte University Deprtment of Microbiology culture collection. The clinicl E. coli isolte ws obtined from ptient with urinry trct infection nd ws provided by

2 2234 CAMPER ET AL. the Bozemn Deconess Hospitl, Bozemn, Mont. Identifiction of ll strins ws performed with API 20E rpid identifiction systems (Anlytb Corp., Plinview, N.Y.). All cultures were stored t -70 C in 20% peptone-40% glycerol. Totl orgnic crbon determintions. Totl orgnic crbon levels were determined for double-glss-distilled wter, finished minerl slts (MS) medium, nd yest extrct solutions prior to ech experiment. The orgnic crbon contents of suspended tubercle mteril, dry tubercle, nd the scrped biofilm used s substrtes were lso mesured. All nlyses were done with n Ocenogrphy Interntionl Corp. Orgnic Crbon Anlyzer (model 0524B-8A-3030). Sufficient replicte determintions were performed for ech nlysis to give cceptble precision in the mesurements. Preprtion of conditioned cultures. All glsswre ws cid wshed nd oven sterilized t 180 C for 3 h. The cidwshing procedure consisted of soking the glsswre in chromic cid for t lest 3 h nd then rinsing it three times with tp wter nd three times with ultrpure wter. MS medium, which consisted of K2HPO4 (7.0 g liter-'), KH2PO4 (3.0 g liter-'), (NH4)2SO4 (1.0 g liter-'), nd MgSO4 7H20 (0.1 g liter-'), ws utoclved prior to use. All medi were prepred with fresh double-glss-distilled wter which consistently contined under 0.8 mg of totl orgnic crbon liter-'. Aliquots of 100 ml of MS medium contining 10 mg of yest extrct (Difco Lbortories, Detroit, Mich.) liter-' were dded to 250-ml Erlenmeyer flsks. The flsks were inoculted with 0.1 ml of the frozen stock cell suspension. Ech flsk ws incubted t 25 C for 24 h, fter which 0.1 ml ws trnsferred to flsk contining 5.0 mg of yest extrct liter-' in MS medium. Following 24-h incubtion period t 25 C, this culture ws diluted with sterile double-glss-distilled wter to deliver finl concentrtion of 102 to 103 cells per ml in the growth experiment test flsks. Growth experiments. A stock solution of yest extrct ws prepred nd diluted with double-glss-distilled wter to provide finl concentrtions of 0.1, 0.25, 0.5, nd 1.0 mg liter-' in 100 ml of MS medium. The yest extrct contined 62% orgnic crbon by weight. The flsks were inoculted with the conditioned coliform cultures. A zero time smple ws removed, nd bcteril numbers were determined in triplicte by the spred plte technique on 10% plte count gr (1/10-strength Difco plte count gr, 1.5% totl gr). Pltes were incubted t 25 C for 24 h, nd the colonies were counted. The flsks were incubted t 25 C nd smpled every 24 h for plte counts until the sttionry phse ws reched. All experiments were performed with triplicte flsks nd were conducted three times. Similr procedures were used to ssess the effect of temperture on the growth of coliforms in the presence of low nutrient concentrtions. Triplicte flsks contining 0.1 or 1.0 mg of yest extrct liter-' were inoculted to finl concentrtion of 102 to 103 coliforms per ml with preconditioned K. pneumonie, E. coli, or Enterobcter erogenes. The test flsks underwent sttionry incubtion t 10, 15, nd 20 C. Growth ws monitored s in the prior experiments until the sttionry phse ws reched. The effect on coliform growth of iron tubercle mteril from drinking wter distribution systems ws exmined. Three flsks contining 100 ml of MS medium plus 0.1 or 1.0 mg of yest extrct liter-' nd 20 mg of pulverized, utoclved, nd dried iron tubercle liter-' were inoculted with K. pneumonie s described bove. The tubercle mteril ws composite of smples removed from the New Hven APPL. ENVIRON. MICROBIOL. distribution system; it contined, on dry weight bsis, 56% iron, 0.02% mngnese, nd 0.004% clcium. Aluminum, mgnesium, silicon, nd totl orgnic crbon concentrtions were below the levels of detection. The flsks were incubted t 25 C for up to 3 weeks. Smples were removed routinely, nd bcteril numbers were determined in triplicte by the spred plte technique. Prior to inocultion of the pltes, the fluid smples were homogenized (Ultr-Turrx; Tekmr Co., Cincinnti, Ohio) t 4 C for 3 min to ensure n even cell distribution in the inoculum. Experiments were lso performed with biofilm mteril s the sole crbon source. The biofilm, consisting primrily of bcteri nd their extrcellulr polymer, ws removed from simulted polyvinyl chloride distribution system operted with New Hven wter (15). The substnce ws utoclved nd dispersed in quntities of 1.0 ml per 100 ml of MS medium. Mesurements of orgnic crbon in the biofilm were lso performed. Triplicte flsks were inoculted with K. pneumonie, incubted, nd enumerted s previously described. The period of most rpid growth ws used to determine the specific growth rte (,u). It ws clculted by the formul p. = (loglo n2 - log10 n,)(2.303)/(t2 - tl), where n2 is the cell number t time 2 (t2) nd n, is the cell number t time 1 (tl). Determintion of growth in drinking wter. Drinking wter smples were collected t sites in the New Hven distribution system where coliforms hd been recovered. The smples were frozen in cid-wshed sterile 1-liter Nlgene bottles (Nlge Co., Rochester, N.Y.) nd shipped to the lbortory. The wter ws thwed nd psteurized t 65 C for 30 min by the method for ssimilble orgnic crbon described by vn der Kooij et l. (14). Chlorine levels were mesured colorimetriclly (Hch Co., Lovelnd, Colo.) nd found to be below the level of detection. Four liquots of 100 ml were dispensed into 250-ml cid-wshed nd bked Erlenmeyer flsks nd inoculted with ech of the four conditioned coliform cultures which hd received n dditionl pssge in MS medium contining 1.0 mg of yest extrct liter-'. The flsks were incubted t 25 C, nd smples were removed dily for enumertion. RESULTS The orgnisms chosen for this study represent four coliform species reported s being most frequently isolted from the wter of the cooperting drinking wter system. K. pneumonie mde up 30.5% of the totl coliforms identified nd ws the most prevlent species. Enterobcter cloce ws the second most common t 21.8%, E. coli ws sixth t 5.3%, nd Enterobcter erogenes ws seventh t 2.1%. All isoltes except the Enterobcter erogenes species used in these experiments exhibited fecl biotype s determined by gs production in EC broth (Difco) t 44.5 C in 24 h. The origin of the lrge numbers of coliforms detected during regrowth or ftergrowth events in distribution systems hs not been clerly defined, lthough it is ssumed tht these coliforms re not the result of breching the disinfection brrier, of cross-connections, or of min breks. Current thinking is tht they re replicting under distribution system conditions in biofilms on pipe wll surfces. Experiments were designed to determine whether coliforms from the system tht were dpted to the oligotrophic conditions typicl of finished drinking wter exhibited higher growth rte thn copiotrophic clinicl isoltes of the sme species. Figure 1 illustrtes four growth curves for the clinicl nd environmentl K. pneumonie strins grown in unsupple-

3 3.5-.,A_ VOL. 57, 1991 GROWTH KINETICS OF DRINKING WATER COLIFORMS 2235 were lso not significntly different from those for Enterobcter cloce grown t the three highest nutrient levels. 6- At ll yest extrct concentrtions, the specific growth rte of 5.5- the K. pneumonie clinicl isolte ws pproximtely hlf 5-/.. 4.5; C Time Oh FIG. 1. Growth of environmentl nd clinicl K. pneumonie isoltes in the presence of two concentrtions of yest extrct t 25 C. Symbols: *, environmentl strin, 1.0 mg liter-1; O, clinicl strin, 1.0 mg liter-1; U, environmentl strin, 0 mg liter-'; + clinicl strin 0 mg liter-1. mented MS medium nd in the sme medium with 1.0 mg of yest extrct liter-1. The distilled wter used in these experiments contined 0.4 mg of orgnic crbon liter-'. The environmentl isolte grown in MS medium without dded crbon incresed in number by 2 logs, wheres the conditioned clinicl strin numbers incresed by less thn 1 log. When grown in the presence of 1.0 mg of yest extrct liter-', the drinking wter isolte numbers incresed by 3 logs in 23 h, wheres the clinicl orgnism numbers incresed by mximum of 2 logs in 48 h. Specific growth rtes for the four environmentl coliform isoltes, their clinicl counterprts, nd the ETEC strin re presented in Tble 1 for the vrious nutrient concentrtions. All of the orgnisms from the distribution system were cpble of mesurble growth in MS medium mde with double-glss-distilled wter nd no dded yest extrct. The distribution system K. pneumonie generlly hd the highest specific growth rte of the four wter isoltes in the presence of ll yest extrct concentrtions bove zero. These rtes TABLE 1. tht of the distribution system culture. The specific growth rte of the E. coli wter isolte ws slightly lower thn tht of the K. pneumonie t ll yest extrct levels. The clinicl E. coli cell numbers did not increse when no yest extrct ws dded, but incresed s the yest extrct levels incresed. At 0.5 nd 1.0 mg of yest extrct liter-', the clinicl strin grew s rpidly s the environmentl isolte. Unexpectedly, the highest specific growth rte for the ETEC strin ws only slightly lower thn tht of the K. pneumonie wter isolte. The Enterobcter erogenes strin hd the lowest growth rte of ll the environmentl isoltes t 0, 0.1, nd 0.25 mg of yest extrct liter-' nd comprble growth rte to the others t the two highest nutrient concentrtions. At greter yest extrct concentrtions, ll four environmentl isoltes hd similr specific growth rtes (,. = 0.30 to 0.22 h-1), with the K. pneumonie nd Enterobcter cloce strins hving the highest rtes. Rnks of the growth rtes for the four orgnisms under the low-nutrient conditions were similr to the reltive bundnces of the isoltes in the distribution system. For exmple, the K. pneumonie drinking wter isolte hd the highest specific growth rte t 0.1 nd ws the most frequently isolted coliform, wheres the Enterobcter erogenes isolte hd the lowest specific growth rte nd ws the lest frequently isolted coliform. The mximl numbers of bcteri produced t ech of the nutrient levels re presented in Tble 2. Of interest is the difference in finl cell concentrtions t the lower yest extrct concentrtions. When no yest extrct ws present, ll of the strins except the clinicl E. coli isolte were cpble of some growth. The environmentl K. pneumonie mg liter-' isolte ttined greter numbers thn the other species t very low nutrient concentrtions (0 nd 0.1 mg liter-'). The ETEC strin hd cell yield identicl with tht of the environmentl isolte t ll concentrtions except 0 mg liter-', when the yield ws greter thn with the environmentl strin. At the higher yest extrct concentrtions, ll strins produced equivlent cell yields. Specific growth rtes of coliforms in vrious concentrtions of yest extrct t 250C Orgnism nd Specific growth rte (,. [h-1]) ± SE for yest extrct concn (mg liter-'): isolte K. pneumonie Wter isolte 0.10 ± ± ± ± ± Clinicl isolte 0.07 ± ± ± ± ± E. coli Wter isolte ± ± ± ± Clinicl isolte NGb 0.08 ± ± ± ± ETEC 0.09 ± ± ± ± ± E. erogenes Wter isolte 0.04 ± ± ± ± ± E. cloce Wter isolte 0.08 ± ± ± ± Men vlue for,. for three mesurements from three replicte experiments. One-wy nlysis of vrince on growth rtes for ll seven isoltes t ech substrte concentrtion demonstrted tht the vlues shown re different t the 0.01 level. b NG, no growth detected.

4 2236 CAMPER ET AL. APPL. ENVIRON. MICROBIOL. TABLE 2. Mximum cell production of coliforms in vrious concentrtions of yest extrct t 250C Orgnism nd Mx cell production (no. per ml) in yest extrct concn (mg liter-'): isolte K. pneumonie Wter isolte 9.5 x x x x X 106 Clinicl isolte 4.9 x X X x x 105 E. coli Wter isolte 2.6 x x 10' 3.1 x x x 106 Clinicl isolte -0.5 x x x x x 105 ETEC 4.3 x x x x x 106 E. erogenes Wter isolte 4.0 x x X x x 106 E. cloce Wter isolte 1.4 x x x x x 105 Decrese in cell number from inoculum. The observtion tht the coliforms isolted from drinking wter were ble to proliferte under conditions of very low orgnic crbon motivted us to exmine coliform growth in 12 unsupplemented drinking wter smples. Of these, four smples contined enough ssimilble orgnic crbon to support growth of t lest one of the coliforms (Tble 3). The two wter smples with the lowest totl orgnic crbon content supported the growth of one coliform ech, the wter smple with n intermedite level (3.02 mg liter-1) permitted growth of two of the coliforms, nd the wter smple contining the highest orgnic crbon level (5.44 mg liter-') permitted growth of ll three coliforms tested. A potentil source of nutrients for bcteri in drinking wter distribution systems my be bcteril products ssocited with biofilm. This possibility ws exmined by using biofilm mteril which hd been obtined from the surfce of polyvinyl chloride pipe system receiving treted drinking wter from filtrtion plnt s the sole crbon source in growth experiments. A crbon-hydrogen-nitrogen elementl nlysis of the utoclved biofilm indicted tht it contined 34.9% crbon, 5.4% hydrogen, nd 5.4% nitrogen. A sufficient mount of this mteril ws dded to MS medium to give totl orgnic crbon concentrtion of 0.5 mg liter-'. The environmentl isolte of K. pneumonie grown in this medium incresed in numbers by pproximtely 1 log over the level for growth without the biofilm mteril. When 20 mg of pulverized iron tubercle liter-' ws dded to flsks contining 0, 0.1, nd 1.0 mg of yest extrct liter-', there ws very little enhncement of growth of the TABLE 3. Orgnic crbon content nd number of environment coliform bcteri' produced in unsupplemented drinking wter Wter smple Totl orgnic No. of cells ml-' for: crbon concn (mg liter-') + SEb K. pneumonie E. erogenes E. coli ± x 105 NGc NG ± 0.28 NG 7.1 x 103 NG ± x X 105 NG ± x x x 105 Men vlue of vible bcteri s determined by plte count from three replictes; initil inocul were less thn 102 ml-l. b Men totl orgnic crbon mesured in triplicte before inocultion. c NG, no growth detected. environmentl K. pneumonie strin when compred with incubtion without tubercle. For exmple, fter 48 h of incubtion t 25 C, 1.6 x 106 bcteri per ml were enumerted from the test flsk contining 1.0 mg of yest extrct liter-' nd 20 mg of tubercle nd 1.4 x 106 bcteri were enumerted from control flsk without tubercle (men vlues for triplicte redings from triplicte smples). Strting cell numbers were ner 102/ml. Specific growth rtes were lso similr for cultures with nd without tubercle (,. = 0.30 nd 0.29 h-1, respectively). The cpbility of the coliforms to grow t low levels of orgnic crbon suggested tht they my lso be cpble of growth t wter tempertures ner those typiclly observed in distribution systems nd tretment fcilities. Tempertures in the New Hven distribution system rnged from 4.8 C in Februry to 21.9 C in August (monthly verge tempertures). Three of the coliforms isolted from wter (K. pneumonie, E. coli, nd Enterobcter erogenes) were incubted t 10, 15, 20, nd 25 C in MS medium plus 0.1 or 1.0 mg of yest extrct liter-'. Specific growth rtes (pu) for ech of the three species t the four tempertures with 0.1 mg of yest extrct liter-' re presented in Fig. 2. The environmentl E. coli isolte demonstrted no increse in cell numbers fter 3 weeks of incubtion t 10 C. The other two coliforms were cpble of prolifertion t the low tempertures documented in drinking wter system. When 0.1 mg of yest extrct liter-' ws used, mximl cell counts of c. 105 bcteri per ml were reched within 5 dys for the K. pneumonie isolte t 10 C nd 10 dys for Enterobcter erogenes. The higher tempertures (15, 20, nd 25 C) yielded c. 105 bcteri per ml within 4 dys for ll three isoltes. At the higher yest extrct concentrtion (1.0 mg liter-) nd higher tempertures, c. 106 bcteri per ml were found fter 2 dys of incubtion. The sme cell numbers were produced t 10 C but only fter some dely (6 dys for K. pneumonie, 5 dys for E. coli, nd 8 dys for Enterobcter erogenes). The specific growth rtes t the higher yest extrct concentrtion were similr to those t 0.1 mg liter-'. DISCUSSION Vrious reserchers hve shown tht coliforms cn proliferte t extremely low crbon levels. Zobell nd Grnt (18), s erly s 1943, reported tht E. coli grew in solutions

5 VOL. 57, 1991 GROWTH KINETICS OF DRINKING WATER COLIFORMS ' 0) 0.-Z t_ O Temperture (CC) FIG. 2. Specific growth rtes of environmentl coliform isoltes s function of incubtion temperture with 0.1 mg of yest extrct liter-'. Symbols: *, K. pneumonie; *, E. coli; +, Enterobcter erogenes. contining 0.1 mg of glucose liter-'. Vn der Kooij et l. demonstrted tht Aeromons hydrophil ws cpble of growing with 0.1 mg of C liter-' s glucose (13). Drinking wter isoltes of this orgnism were ble to rech yields of up to 5 x 105 CFU ml-' in the presence of 0.5 mg of peptone liter-' nd 7 x 104 CFU ml-' in distilled wter contining 4.2 mg of dissolved orgnic crbon liter-', with corresponding mximum growth rtes of 0.3 nd 0.08 h-' (11). However, growth kinetics nd cell yields re lcking for coliform isoltes from drinking wter, especilly those of the fecl biotype. The specific growth rtes for the coliform most frequently isolted from the New Hven distribution system, K. pneumonie, rnged from low of 0.10 h-1 in unsupplemented glss-distilled wter'to 0.29 h-1 t 1.0 mg of yest extrct liter-1 t 25 C. These specific growth rtes, on verge, exceeded those of the other coliforms tested, s did cell yields. The specific growth rte of the clinicl isolte ws pproximtely hlf tht of the environmentl strin. In ddition, the specific growth rte of the drinking wter K. pneumonie isolte incresed in ner-liner fshion with incresing temperture when the orgnism ws grown on 0.1 TABLE 4. mg of yest extrct liter-'; the growth rtes were lso greter thn those of the other two coliforms. These dt indicte tht this K. pneumonie isolte is dpted to oligotrophic conditions nd tempertures representtive of distribution systems nd my explin why it ws the most frequently isolted coliform. Of prticulr interest nd concern re the results obtined with the three E. coli strins. The growth rte t the vrious crbon concentrtions for the environmentl strin exceeded tht of the clinicl isolte, but both were surpssed by tht of the ETEC strin. The yields for the environmentl strin nd the ETEC strin were identicl under the sme conditions. Although these results re not definitive, becuse the ETEC strin hd been pssed for severl genertions in the lbortory, they indicte tht the ETEC strin, wterborne pthogen, my lso be ble to proliferte t low nutrient concentrtions. The growth of the environmentl isolte under the sme conditions would yield similr results in totl coliform test, but the public helth significnce would be fr lower. A summry of the kinetic dt nd yields clculted for ll the strins grown on yest extrct is presented in Tble 4. The reported vlues for yield determined s milligrms of cell crbon per milligrm of substrte crbon re n order of mgnitude lower thn those usully expected for copiotrophic growth (0.4 to 0.5 mg of cell C/mg of substrte C). Although it is possible tht this difference is due to computtionl ssumptions for cell volume, dry mss per cell, nd percentge of dry mss s crbon, we speculte tht these low yields re the result of significnt energy expenditure by the cells in procuring the limited substrte vilble in solution. When exmining yields obtined on crbon bsis nd on cell bsis, we find tht the yields for the clinicl isoltes nd Enterobcter cloce re significntly lower thn those for the environmentl strins. The ETEC yields re similr to those for the K. pneumonie nd E. coli wter isoltes. Vlues for Ks, s determined by nonliner regression, re ll well below 1 mg liter-1, which is indictive of substrte uptke systems cpble of operting under low nutrient (<1.0-mg liter-') conditions. Ks vlues for the environmentl K. pneumonie nd E. coli strins"nd the ETEC strin re hlf or less thn those for the 'linicl strins. The clculted mximum growth rtes (ILmx) rnged from 0.16 to 0.37 h-1, bout hlf those observed for coli- Cell yield nd kinetic prmeters for environmentl nd clinicl coliforms Substrte co- Mximum SE for Cell yield on SE for cr- Concel- Cell yield on cell SE for cell yield, Isolte efficient, K. growth rte, growth rte, crbon bsis, bon yield, or fiin for bsis, Y (cells v, (cells mg of (mg liter-') PLmx (h-')., (h1-) Y(mg mg-') (mg mg-) yield, r2 mg of C-') C-) K. pneumonie Wter isolte x x x 107 Clinicl isolte x x x 106 E. coli Wter isolte x x x 107 Clinicl isolte x x x 107 ETEC x x x 107 E. erogenes x x x 107 E. cloce x x x 107 L.mx nd K. were determined by nonliner regression. Cell number yields were determined by liner regression of the cell number with substrte crbon. Cell mss yields were bsed on g (dry mss) per cell, 40% crbon within dry mss. Yest extrct ws determined to contin 62% crbon.

6 2238 CAMPER ET AL. forms under idel growth conditions (c h-'). These dt lso support the hypothesis tht the environmentl coliforms, especilly the K. pneumonie nd E. coli isoltes, s well s the ETEC strin, re dpted to growth under oligotrophic conditions. The source of crbon in distribution system wter for growth of these orgnisms is lso of concern. Severl potentil nutrient sources were tested in ddition to the yest extrct. These included utoclved biofilm, crushed tubercle mteril, nd the ssimilble orgnic crbon frction of treted drinking wter. K. pneumonie ws grown in the presence of the biofilm t concentrtion of 0.5 mg of totl orgnic crbon liter-1. When compred with the yest extrct concentrtion most similr in crbon concentrtion to this vlue (1.0 mg liter-1), similr increses of 1 log over the vlue for unsupplemented minerl slts in cell yields were seen. Biofilm itself ppered to be suitble crbon source for the prolifertion of this coliform. In our experiments, crushed tubercle mteril did not enhnce the growth of the orgnism. These results disgree with those of Victoreen (16, 17), Mrtin et l. (8), nd Allen et l. (1), ll of whom showed tht tubercle mterils or their soluble frction cused n increse in coliform numbers. The difference my be due to the coliforms chosen or the chemicl composition of the tubercle mteril used. There hs recently been significnt interest in the mount of ssimilble orgnic crbon present in drinking wter nd its effect on coliform growth nd distribution system opertions (6). Vn der Kooij et l. (14) developed the technique in which specific Pseudomons strin ws grown in drinking wter nd the resulting increse in cell numbers ws converted to levels of cette utiliztion. A similr procedure ws used in this study with the drinking wter coliforms s the inoculum. Four unsupplemented drinking wter smples were obtined during period of norml system opertion nd were inoculted with K. pneumonie, E. coli, nd Enterobcter erogenes. Orgnic crbon concentrtions in these wter smples vried from 1.44 to 5.44 mg liter-'. All of the wter smples supported the growth of t lest one of the coliforms when incubted t 25TC. K. pneumonie nd Enterobcter erogenes ech grew in three of the wter smples, nd the E. coli isolte grew in one. The mximum cell yields were pproximtely i05 cells for ech orgnism. The wter smple with the gretest orgnic crbon concentrtion llowed for prolifertion of ll three coliforms. These results show tht sufficient nutrients exist in the distribution wter itself to support the growth of coliforms nd tht environmentl isoltes my be superior indictor orgnisms for determining the mount of ssimilble orgnic crbon. Clerly, the results support the hypothesis tht growth of environmentl coliform isoltes is possible under environmentl conditions typiclly found in distribution systems. Since these bcteri re the system nuisnce orgnisms nd pper to thrive on the conditions therein, it is only resonble to use them to determine the bility of the wter to support their growth. Considering tht plnktonic growth ws demonstrted under low-nutrient nd low-temperture conditions, biofilm growth is even more likely, especilly since biofilm system conditions hve been shown to support coliform growth under nutrient limittion (4, 7). Becuse the environmentl coliforms tested in these studies were isolted from distribution system nd becuse they re cpble of growth t low tempertures nd low nutrient levels which occur in distribution systems, these coliforms cn be component of the biofilm consorti. The public helth significnce of these results is tht fecl coliforms were cpble of prolifertion, even t low tempertures nd under poor nutrient conditions. The tendency to consider these orgnisms of limited public helth significnce, even-though they re fecl coliforms, must be cutioned ginst since n ETEC strin ws lso ble to grow. ACKNOWLEDGMENTS We thnk the stff of the South Centrl Connecticut Regionl Wter Authority, especilly Drrell Smith, for their ssistnce nd encourgement. We lso grtefully cknowledge the contribution Brry Pyle nd the technicl ssistnce of Theres Rmirez nd Tom Guz, Deprtment of Microbiology, nd Ewout vn der Wende, Center for Interfcil Microbil Process Engineering. This project ws funded in prt by the Americn Wter Works Assocition Reserch Foundtion nd the South Centrl Connecticut Regionl Wter Authority nd prtly by the Ntionl Science Foundtion Engineering Reserch Center Progrm nd the Center for Interfcil Microbil Process Engineering Industril Assocites. REFERENCES APPL. ENVIRON. MICROBIOL. 1. Allen, M. J., nd E. E. Geldreich Distribution line sediments nd bcteril regrowth, p In Americn Wter Works Assocition Wter Qulity Technology Conference, Phildelphi. Americn Wter Works Reserch Foundtion, Denver. 2. Bouwer, E. J., nd P. B. Crowe Biologicl processes in drinking wter. J. Am. Wter Works Assoc. 80: Centers for Disese Control Detection of elevted levels of coliform bcteri in public wter supply. Morbid. Mortl. Weekly Rep. 34: Dvies, D. G., nd G. A. McFeters Growth nd comprtive physiology of Klebsiell oxytoc ttched to GAC in liquid medi. Microb. Ecol. 15: Edberg, S. C., V. Piscitelli, nd M. Crter Phenotypic chrcteristics of coliform nd noncoliform bcteri from public wter supply compred with regionl nd ntionl clinicl species. Appl. Environ. Microbiol. 52: Huck, P. M Mesurement of biodegrdble orgnic mtter nd bcteril growth potentil in drinking wter. J. Am. Wter Works Assoc. 82: LeChevllier, M. W., T. M. Bbcock, nd R. G. Lee Exmintion nd chrcteriztion of distribution system biofilms. Appl. Environ. Microbiol. 53: Mrtin, R. S., W. H. Gtes, R. S. Tobin, D. Grnthm, R. Sumrh, R. Wolfe, nd P. Forestll Fctors ffecting coliform bcteri grqwth in distribution systems. J. Am. Wter Works Assoc. 65: Servis, P., G. Billen, nd M. C. Hscoet Determintion of the biodegrdble frction of dissolved orgnic mtter in wter. Wter Res. 21: Symons, J. M., T. A. Bellr, J. K. Crswell, J. DeMrco, K. L. Kropp, G. G. Robeck, D. R. Seeger, C. J. Slocum, B. L. Smith, nd A. A. Stevens Nturl orgnics reconnissnce survey for hlogented orgnics. J. Am. Wter Works Assoc. 67: vn der Kooij, D., nd W. A. M. Hijnen Nutritionl verstility nd growth kinetics of n Aeromons hydrophil strin isolted from drinking wter. Appl. Environ. Microbiol. 54: vn der Kooij, D., J. P. Ornje, nd W. A. M. Hijpen Growth of Pseudomons eruginos in tp wter in reltion to utiliztion of substrtes t concentrtions of few microgrms per liter. Appl. Environ. Microbiol. 44: vn der Kooij, D., A. Visser, nd W. A. M. Hijnen Growth of Aeromons hydrophil t low concentrtions of substrtes dded to tp wter. Appl. Environ. Microbiol. 39: vn der Kooij, D., A. Visser, nd W. A. M. Hijnen

7 VOL. 57, 1991 GROWTH KINETICS OF DRINKING WATER COLIFORMS 2239 Determining the concentrtion of esily ssimilble orgnic crbon in drinking wter. J. Am. Wter Works Assoc. 74: vn der Wende, E., W. G. Chrcklis, nd D. B. Smith Biofilms nd bcteril drinking wter qulity. Wter Res. 23: Victoreen, H. T The stimultion of coliform growth by hrd nd soft wter min deposits. In Americn Wter Works Assocition Wter Qulity Technology Conference, Mimi Bech, Fl. Americn Wter Works Reserch Foundtion, Denver. 17. Victoreen, H. T The role of rust in coliform regrowth, p In Americn Wter Works Assocition Wter Qulity Technology Conference, Denver. Americn Wter Works Reserch Foundtion, Denver. 18. Zobell, C. E., nd C. W. Grnt Bcteril utiliztion of low concentrtions of orgnic mtter. J. Bcteriol. 45: